Synthesis and antitumor activity evaluation in vitro of 4-aminoquinazoline derivatives containing 1,3,4-thiadiazole

In order to search for new antitumor drugs with high efficiency and low toxicity, a series of novel 4-aminoquinazoline derivatives containing 1,3,4-thiadiazole group were designed, synthesized and evaluated for antiproliferative activity against four human cancer cell lines (H1975, PC-3, MCF-7 and HGC-27) using MTT assay in vitro. Among them, compound N-(5-((3,5-dichlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)-2-((4-(phenylamino)quinazolin-2-yl)thio)acetamide (15o) showed good anti-tumor proliferation activity against four tested cancer cell lines, with IC50 values of 1.96 ± 0.15 μM to PC-3 cell. The antitumor activity was significantly better than that of gefitinib. Further mechanism studies showed that compound 15o inhibited the migration ability of PC-3 tumor cells in a concentration dependent and time-dependent manner, and blocked the cell cycle in S phase. At the same time, cell cloning experiment further proved that compound 15o significantly inhibited the colony formation of PC-3 cells, The inhibition rate was as high as 92% at 2.0 μM. Graphical abstract Graphical abstract


Introduction
Malignant tumor is a tissue with abnormal physiological function formed by excessive proliferation or abnormal differentiation of tumor cells. At present, the clinical treatment methods of malignant tumors include surgical treatment, radiotherapy, chemotherapy, immunotherapy and targeted therapy, among which chemotherapy is still an effective method for the treatment of malignant tumors [1][2][3][4]. However, the adverse side effect and the development of resistance to traditional anticancer drugs call for an urgent exploration of new anticancer agents [5,6].

Chemistry
The synthetic strategy to prepare the target compounds was depicted in Fig. 2. First dissolved KOH in water, and then dissolved 5-amino-1,3,4-thiadiazole-2-thiol in the aqueous solution of potassium hydroxide. After the system became clear, substituted benzyl chloride compounds are added to the system and reacted at room temperature for 8 h to obtain compounds (9a-o). Then, compounds (10a-o) were acquired from the reaction of compound (9a-o) with chloroacetyl chloride at room temperature for 3.5 h. Next, Aniline, triethylamine and carbon disulfide were added to acetone solution and reacted at room temperature for two hours to obtain a yellow solid. The solid obtained by suction filtration was compound (12). Then, compound (13) was acquired from the reaction of compound (12) with triphosgene in chloroform at room temperature for 0.5 h, that was further reacted with o-anthranilonitrile in pyridine at 100°C for 8 h to get compound (14). Finally, dissolved compound (14) in the aqueous solution of potassium hydroxide. After the system became clear, dissolved compounds (10a-o) with 1,4-dioxane and added it to the above system reacted at 70°C for two hours to obtain white solid, and then filter to obtain compounds (15ao). The structure of target compounds were confirmed by 1 H NMR, 13 C NMR, IR and HRMS.

MTT assay
The in vitro antiproliferative activities of all the prepared compounds (15a-o) were evaluated against H1975 (Human lung cancer cell line), PC-3 (Human prostate cancer cell line), MCF-7 (Human breast cancer cell line) and HGC-27 (Human gastric carcinoma cell line) by MTT assay. Gefitinib was selected as positive reference drugs. The IC 50 values (concentration required to achieve 50% inhibition of  Table 1. The synthetic strategy to prepare the target compounds was depicted in Fig. 2. First dissolved KOH in water, and then dissolved 5-amino-1,3,4-thiadiazole-2thiol in the aqueous solution of potassium hydroxide. After the system became clear, substituted benzyl chloride compounds are added to the system and reacted at room temperature for 8 h to obtain compounds (9a-o). Then, compounds (10a-o) were acquired from the reaction of compound (9a-o) with chloroacetyl chloride at room temperature for 3.5 h. Next, Aniline, triethylamine and carbon disulfide were added to acetone solution and reacted at room temperature for two hours to obtain a yellow solid. The solid obtained by suction filtration was compound (12). Then, compound (13) was acquired from the reaction of compound (12) with triphosgene in chloroform at room temperature for 0.5 h, that was further reacted with o-anthranilonitrile in pyridine at 100°C for 8 h to get compound (14). Finally, dissolved compound (14) in the aqueous solution of potassium hydroxide. After the system became clear, dissolved compounds (10a-o) with 1,4-dioxane and added it to the above system reacted at 70°C for two hours to obtain white solid, and then filter to obtain compounds (15a-o). The structure of target compounds were confirmed by 1 H NMR, 13 C NMR, IR and HRMS.

Effect of compound 15o against normal human cell lines
To further investigate the toxicity of these compounds, compound 15o which had best antiproliferative activities against tumour cell lines in Table 1 was picked out to further determine its effects on normal human gastric epithelial cell (GES-1) and human normal esophageal epithelial cells (HEEC). As shown in Table 2, it showed weak or no toxicity against GES-1 and HEEC.

Cell cycle arrest by compound 15o
Compound 15o was chosen to further evaluate its possible anticancer mechanism of action against PC-3 cell based on the above results. After 24 h treatment with compound 15o, PC-3 cells were arrested in S phase (Fig. 3). With the increase of compound 15o concentration, the percentage of

Cell migration inhibition arrest by compound 15o
As shown in Fig. 4, with the extension of time and the increase of compound 15o concentration, the cell migration area gradually decreases, indicating that the migration ability of PC-3 cells decreases, and compound 15o inhibits the migration of PC-3 cells in a time-dependent and concentration-dependent manner.

Cell clone arrest by compound 15o
As shown in Fig. 5, compared with the control group, with the increase of compound 15o concentration, the number of cell clone formation decreased and the cell community decreased significantly, indicating that compound 15o significantly inhibited the colony formation of PC-3 cells, The inhibition rate was as high as 92% at 2.0 μM.

Conclusion
In this work, a series of novel 4-aminoquinazoline derivatives bearing 1,3,4-thiadiazole group were designed, synthesized and evaluated for antiproliferative activity against four human cancer cell lines (H1975, PC-3, MCF-7, and HGC-27). The results suggested that most compounds had potent anti-proliferative activity. Especially, the compound (15o) exhibited the best antiproliferative activity against H1975, PC-3, MCF-7, and HGC-27 cell lines. Further studies showed that compound 15o could inhibit the cloning and migration of PC-3 cell and block the cell cycle in S phase. The above facts illustrated that this work would have remarkable implications for further design and development of a possible antitumor agent.

Materials
Reagents and solvents were purchased from commercial sources and were used without further purification. High resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass spectrometer by electrospray ionizaton (ESI). 1

Synthesis of compounds
Compounds (9-10) [23,24] and compounds (12)(13)(14) [25] were synthesized according to the published literature. Potassium hydroxide (4.96 mmol) was dissolved in water (15 ml) then compound (8) (4.50 mmol) was added. Stirred at room temperature until the solid disappeared. Different benzyl chloride compounds were added to the system and reacted at room temperature for 8 h to obtain large amount of white precipitation. Then compounds (9a-o) were obtained by filtration and drying. The compounds 9a-o (2.33 mmol) were added to 25 ml round bottom flask, then 8 ml of 1,4-dioxane was added to dissolve them, then chloroacetyl chloride (2.79 mmol) was slowly added to the system and reacted at room temperature for 3.5 h. After the monitored reaction, poured the reaction system into the ice water mixture to precipitate a large amount of solid, filtered and dried to obtain the compounds (10a-o). Aniline (5.37 mmol) was added to a 25 ml round bottomed flask, dissolved in 5 ml acetone, and then triethylamine (26.84 mmol) and carbon disulfide (26.84 mmol) were added in turn. After reaction at room temperature for 2 h, a large amount of precipitation is generated, filtered and dried to obtain compound (12). Triphosgene (0.98 mmol) was added to a 25 ml round bottom flask and dissolved by adding 6 ml of chloroform. Then compound (12) (1.97 mmol) was added to the above system and reacted at room temperature for 0.5 h. Compound (13) was separated by column chromatography after the reaction was detected by thin layer chromatography. O-aminobenzonitrile (6.77 mmol) was added to a 50 ml round bottom flask, followed by 10 ml of pyridine and compound 13 (8.13 mmol) at 100°C for 8 h. After the reaction was detected by TLC, 10 ml of mixed solution of petroleum ether and ethanol was added to the reaction system to produce a large amount of precipitation. Compound (14) was obtained by filtration and drying. General procedure for synthesis of derivatives (15a-o). Potassium hydroxide (1.18 mmol) was dissolved in water (20 ml) then compound (14) (0.79 mmol) was added. When the system became transparent, compounds (10a-o) (0.75 mmol) were dissolved in dioxane (3 ml) and added to the reaction system, the mixture was heated to 70°C for 2 h, a large amount of white precipitate was precipitated out. After the reaction was completed, filtered (the filter cake was washed with water and dioxane for three times), dried and then crude products were purified by column chromatography to obtain compounds (15a-o).

Evaluation of antiproliferative activity
Cells in the logarithmic growth phase cells at 3000-5000 cells/well were seeded in 96-well plates for 24 h. Then the cells were treated in triplicate with various concentrations of each compound at 37°C in the humidified 5% CO 2 atmosphere for 72 h. And added MTT to each well at a final concentration of 5 mg/ml. After incubated for 4 h in 37°C, the medium was aspirated. Add 150 μL DMSO to each well to dissolve formazan and the plate was shaken on a shaker for 10 min. The OD vaules was measured by microplate reader at a wavelength of 490 nm and The IC 50 values were obtained using SPSS 20.0 software. Results were Mean ± SD of three independent experiments.

Cell cycle distribution assay
The PC-3 cells in logarithmic growth phase were divided into 2 × 10 5 / well were inoculated in 6-well plates and treated overnight at 37°C and 5% CO 2 . The concentrations were 0 (blank control group), 0.5, 1.0 and 2.0 μM. PC-3 cells were treated with compound 15o and treated for 24 h at 37°C and 5% CO 2 .Then cells were harvested and fixed with 70% ethanol for 8 h at 4°C. The fixed cells were washed and resuspended using phosphate buffer saline (PBS) containing 50 mg/mL propidium iodide (PI) and 10 mg/mL RNaseA. Then cell suspension was incubated for 40 min in a dark place at room temperature. After that, samples were analyzed for DNA content with flow cytometry. (Becton, Dickinson and Company, NJ).

Cell migration experiment
The PC-3 cells in logarithmic growth phase were divided into 4 × 10 5 /well were inoculated in 6-well plates and treated overnight at 37°C and 5% CO 2 . When the cell density reached 85%, The 200 μL gun head evenly marks the "cross" at the bottom of the 6-well plates. The cells were treated with concentrations were 0 (blank control group), 0.5, 1.0 and 2.0 μM, and photographed at the same position of each well with a microscope at 0, 12 and 24 h to record the trend of cell migration.
Cell clone arrest PC-3 cells in logarithmic growth stage were inoculated in 6-well plates at 1000/ well, and cultured overnight at 37°C and 5% CO 2 . PC-3 cells were treated with compound 15o at concentrations of 0 (blank control group), 0.5, 1.0 and 2.0 μM under the condition of 37°C and 5% CO 2 for 8 days. When the cell community visible to the naked eye appeared at the bottom of the 6-well plate, the culture was terminated. The cells were fixed at 4°C with 4% paraformaldehyde for 0.5 h, and stained with 0.1% crystal violet for 20 min.